organic acid-tolerant microorganisms and methods of using same. The organic acid-tolerant microorganisms comprise modifications that reduce or ablate acsa activity or acsa homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid. Further modifications to the microorganisms such as increasing expression of malonyl-CoA reductase and/or acetyl-CoA carboxylase provide or increase the ability of the microorganisms to produce 3HP. Methods of generating an organic acid with the modified microorganisms are provided. Methods of using acsa or homologs thereof as counter-selectable markers include replacing acsa or homologs thereof in cells with genes of interest and selecting for the cells comprising the genes of interest with amounts of organic acids effective to inhibit growth of cells harboring acsa or the homologs.
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16. A method of using acsa or homolog thereof as a counter-selectable marker comprising:
replacing an acsa or homolog thereof in a bacterium with a gene of interest; and
selecting for the bacterium comprising the gene of interest with an amount of an organic acid effective to inhibit growth of bacteria harboring a functional acsa gene or homolog thereof.
1. An organic acid-tolerant microorganism comprising a modification that reduces or ablates acsa activity or acsa homolog activity in the microorganism, wherein tolerance to an organic acid selected from the group consisting of 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid is increased compared to a corresponding microorganism not comprising the modification, wherein the microorganism is a bacterium.
3. The microorganism of
5. The microorganism of
6. The microorganism of
7. The microorganism of
9. The microorganism of
11. A method of producing an organic acid comprising culturing a microorganism as recited in
12. The microorganism of
13. The microorganism of
14. The microbial culture of
15. The method of
17. The method of
18. The method of
19. The method of
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This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application 61/647,001 filed May 15, 2012, the entirety of which is incorporated herein by reference.
This invention was made with government support under FA9550-11-1-0038 awarded by the USAF/AFOSR and DE-FC02-07ER64494 awarded by the US Department of Energy. The government has certain rights in the invention.
The present invention relates to organic acid-tolerant microorganisms and uses thereof for producing organic acids.
Production of industrially useful chemicals has conventionally focused on the use of petroleum-like compounds as starting materials. However, various factors have increased interest in the production of such chemicals through microorganism-mediated bioconversion of biomass and other renewable resources.
Accordingly, the U.S. Department of Energy (DOE) recently identified several “building block” chemicals to be produced via microorganism consumption of biomass. The identified chemicals include 1,4 succinic acid, fumaric and malic acids, 2,5 furan dicarboxylic acid, 3-hydroxypropionic acid (3HP), aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol. These chemicals can be converted to high-value, bio-based chemicals or materials.
As an example, 3HP can be readily transformed into a variety of commodity chemicals such as acrylic acid, methyl acrylate, and 1,3-propanediol. These commodity chemicals represent a multi-billion dollar a year industry and are used in the production of plastics, coatings, and fibers. U.S. demand for acrylic acid in particular is growing, exceeding 1×109 kg/year. The current means of synthesizing acrylic acid include oxidation of propylene. A thermodynamically favorable pathway for microbial production of acrylic acid has not been identified.
One hurdle facing the microbial production of industrially useful chemicals is that many, including 3HP, are toxic to the microbes capable of producing them. Recently, efforts have been made not only to increase microbial output of the chemicals but also to increase microbial tolerance to the chemicals. Some of these efforts have focused on the production of 3HP in the heterotrophic microbe Escherichia coli. See, e.g., U.S. Pat. No. 8,048,624 to Lynch, U.S. Pub. 2011/0125118 to Lynch, U.S. Pub. 2010/0210017 to Gill et al., and Warnecke et al. Metabolic Engineering (2010) 12:241-250.
While focusing on chemical production in heterotrophic microorganisms is a valuable strategy, a potential problem is the availability of carbon and energy sources such as food-based commodities and/or sugars derived from lignocellulosic biomass. An attractive alternative is to use phototrophic microorganisms, such as cyanobacteria. These microorganisms can produce chemical products from CO2 and light energy without relying on consumption of higher-value carbon sources that can be used for other purposes, such as producing food, fuel, or other certain chemicals.
There is a need for microorganisms capable of producing high yields of industrially useful chemicals and having increased tolerance against those chemicals. There is also a need for microorganisms that use non-food-based feedstock in such production.
The present invention addresses the aforementioned needs by providing microorganisms with increased tolerance to organic acids. The present invention also provides microorganisms genetically modified to produce organic acids. Methods of producing organic acids with the microorganisms described herein are also provided.
Some versions of the invention provide an organic acid-tolerant microorganism comprising a modification that reduces or ablates AcsA activity or AcsA homolog activity in the microorganism, wherein tolerance to an organic acid selected from the group consisting of 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid is increased compared to a corresponding microorganism not comprising the modification.
The modification is preferably a genetic modification. The genetic modification is preferably a genetic modification other than or in addition to one resulting in a W49L substitution in AcsA or a corresponding substitution in an AcsA homolog.
The microorganism is preferably a bacterium, more preferably a cyanobacterium, and most preferably a cyanobacterium selected from the group consisting of Synechococcus sp., Prochlorococcus sp., Synechocystis sp., and Nostoc sp.
The tolerance to the organic acid is preferably increased at least about 25-fold in the microorganism of the invention compared to the corresponding microorganism.
The tolerance to the organic acid may include a minimum inhibitory concentration (MIC) of at least about 10 mM to acrylic acid, an MIC of at least about 100 mM to 3HP, and an MIC of at least about 200 mM to propionic acid.
In preferred versions of the invention, the microorganism is capable of producing 3HP.
The microorganism may include at least one recombinant nucleic acid configured to overexpress a 3HP pathway enzyme. The at least one recombinant nucleic acid encoding the 3HP pathway enzyme may include a malonyl-CoA reductase gene, such as the malonyl-CoA reductase gene derived from Chloroflexus aurantiacus. The at least one recombinant nucleic acid encoding the 3HP pathway enzyme may additionally or alternatively include an acetyl-CoA carboxylase gene. The recombinant nucleic acids may be heterologous or may comprise heterologous elements.
Some versions of the invention provide a microbial culture comprising a microorganism as described herein and an amount of an organic acid. The amount of the organic acid may be selected from the group consisting of at least about 10 mM acrylic acid, at least about 100 mM 3HP, and at least about 200 mM propionic acid.
Further versions of the invention provide a method of producing an organic acid comprising culturing a microorganism as described herein in the presence of an amount of an organic acid. The organic acid may be selected from the group consisting of 3HP, acrylic acid, and propionic acid. The amount of the organic acid may be selected from the group consisting of at least about 10 mM acrylic acid, at least about 100 mM 3HP, and at least about 200 mM propionic acid.
Another version of the invention includes methods of using acsA or homolog thereof as a counter-selectable marker. One method includes replacing an acsA or homolog thereof in a cell with a gene of interest and selecting for the cell comprising the gene of interest with an amount of an organic acid effective to inhibit growth of cells harboring a functional acsA gene or homolog thereof. The replacing preferably occurs through homologous recombination. The acsA or homolog thereof is preferably an acsA gene with at least one silent nucleic acid mutation that reduces background mutation frequency. The at least one silent nucleic acid mutation is preferably selected from the group consisting of T144C and G150C in acsA from Synechococcus sp. PCC 7002. The organic acid is preferably acrylate. The cell may comprise any cell in which acsA or a homolog thereof confers sensitivity to organic acids. Such a cell may include a Synechococcus sp. cell or a cell from any microorganism described herein, known in the art, or later discovered that harbors an acsA homolog. The selecting preferably results in the cell being homozygous for the gene of interest.
The objects and advantages of the invention will appear more fully from the following detailed description of the preferred embodiment of the invention made in conjunction with the accompanying drawings.
One version of the invention includes a microorganism wherein an acsA gene product or homolog thereof is functionally deleted. The acsA gene product (AcsA) and homologs thereof are acetyl-CoA synthetases classified under Enzyme Commission (EC) number 6.2.1.1. Other names for these acetyl-CoA synthetases include “acetate-CoA ligases,” “acetyl-CoA ligases,” and “acyl-activating enzymes.”
“Functional deletion” or its grammatical equivalents refers to any modification to a microorganism that ablates, reduces, inhibits, or otherwise disrupts production of a gene product, renders the gene product non-functional, or otherwise reduces or ablates the gene product's activity. “Gene product” refers to a protein or polypeptide encoded and produced by a particular gene. In some versions of the invention, “functionally deleted acsA gene product or homolog thereof” means that the acsA gene is mutated to an extent that an acsA gene product or homolog thereof is not produced at all.
One of ordinary skill in the art will appreciate that there are many well-known ways to functionally delete a gene product. For example, functional deletion can be accomplished by introducing one or more genetic modifications. As used herein, “genetic modifications” refer to any differences in the nucleic acid composition of a cell, whether in the cell's native chromosome or in endogenous or exogenous non-chromosomal plasmids harbored within the cell. Examples of genetic modifications that may result in a functionally deleted gene product include but are not limited to mutations, partial or complete deletions, insertions, or other variations to a coding sequence or a sequence controlling the transcription or translation of a coding sequence; placing a coding sequence under the control of a less active promoter; and expressing ribozymes or antisense sequences that target the mRNA of the gene of interest, etc. In some versions, a gene or coding sequence can be replaced with a selection marker or screenable marker. Various methods for introducing the genetic modifications described above are well known in the art and include homologous recombination, among other mechanisms. See, e.g., Green et al., Molecular Cloning: A laboratory manual, 4th ed., Cold Spring Harbor Laboratory Press (2012) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2001). Various other genetic modifications that functionally delete a gene product are described in the examples below. Functional deletion can also be accomplished by inhibiting the activity of the gene product, for example, by chemically inhibiting a gene product with a small molecule inhibitor, by expressing a protein that interferes with the activity of the gene product, or by other means.
In certain versions of the invention, the functionally deleted gene product may have less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or about 0% of the activity of the non-functionally deleted gene product.
In certain versions of the invention, a cell with a functionally deleted gene product may have less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or about 0% of the activity of the gene product compared to a cell with the non-functionally deleted gene product.
In certain versions of the invention, the functionally deleted gene product may be expressed at an amount less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or about 0% of the amount of the non-functionally deleted gene product.
In certain versions of the invention, the functionally deleted gene product may result from a genetic modification in which at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or more nonsynonymous substitutions are present in the gene or coding sequence of the gene product.
In certain versions of the invention, the functionally deleted gene product may result from a genetic modification in which at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or more bases are inserted in the gene or coding sequence of the gene product.
In certain versions of the invention, the functionally deleted gene product may result from a genetic modification in which at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the gene product's gene or coding sequence is deleted or mutated.
In certain versions of the invention, the functionally deleted gene product may result from a genetic modification in which at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a promoter driving expression of the gene product is deleted or mutated.
In certain versions of the invention, the functionally deleted gene product may result from a genetic modification in which at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of an enhancer controlling transcription of the gene product's gene is deleted or mutated.
In certain versions of the invention, the functionally deleted gene product may result from a genetic modification in which at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a sequence controlling translation of gene product's mRNA is deleted or mutated.
In certain versions of the invention, the decreased activity or expression of the functionally deleted gene product is determined with respect to the activity or expression of the gene product in its unaltered state as found in nature. In certain versions of the invention, the decreased activity or expression of the functionally deleted gene product is determined with respect to the activity or expression of the gene product in its form in a corresponding microorganism. In certain versions, the genetic modifications giving rise to a functionally deleted gene product are determined with respect to the gene in its unaltered state as found in nature. In certain versions, the genetic modifications giving rise to a functionally deleted gene product are determined with respect to the gene in its form in a corresponding microorganism.
Some versions of the invention include a plurality of microorganisms, wherein greater than about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more of the plurality of microorganisms comprise a functionally deleted acsA gene product or homolog thereof. In some versions, the plurality of microorganisms is a microbial culture.
Genetic modifications that can be introduced into the acsA gene or homologs thereof to functionally delete the acsA gene product or homologs thereof, such as generating acsA knockouts, are described in the examples below.
The acsA gene is an acetyl-CoA synthetase gene in the exemplary cyanobacterium Synechococcus sp. PCC 7002, the coding sequence of which can be found in GenBank under accession number NC—010475.1 and is as follows:
(SEQ ID NO: 1)
atgtccgaac aaaacattga atccatcctc caggagcagc
gccttttttc gcctgcacca gactttgctg ccgaggccca
gatcaagagc ttagaccagt accaagccct ctacgaccgg
gcgaaaaatg accccgaagg cttttggggg gaactcgccg
aacaggaatt ggaatggttt gagaaatggg acaaggtgct
cgattggcaa ccgcccttcg ccaaatggtt tgtcaacggg
aaaattaaca tttcctacaa ttgcctcgac cgtcatctca
aaacctggcg caaaaataaa gccgccctca tctgggaagg
ggaacccggt gactcccgta ccctcaccta tgcccagcta
caccacgagg tctgccagtt tgccaatgcg atgaaaaagt
tgggcgtcaa aaaaggcgat cgcgtcggga tttatatgcc
aatgatcccg gaagccgtcg ttgccctcct cgcctgtgcc
cgcattggtg cgccccatac ggtgatattt ggtggcttta
gtgccgaagc cctccgcagt cgcctcgaag acgctgaagc
caaactggtg atcaccgccg acgggggctt ccgcaaagat
aaagcggtac ccctcaagga tcaagtagat gcggcgatcg
ccgatcacca tgcccccagc gttgagaatg ttttggtcgt
tcaacgcacc aaagagcctg tccacatgga agccgggcgg
gatcactggt ggcatgattt gcaaaaagaa gtctccgctg
actgtcccgc cgagccgatg gatgccgaag atatgctctt
catcctctat accagcggca ccacgggtaa acccaagggc
gttgtccaca ctacgggcgg ttataatctc tacacccata
taacgaccaa gtggatcttt gatctcaaag atgatgacgt
gtattggtgt ggtgctgatg tgggttggat caccggccac
agttacatta cctatggccc tctatctaac ggggcaacgg
tcttaatgta tgaaggcgca ccccgtccgt ctaatcccgg
ttgctattgg gaaattattc aaaaatatgg tgtcaccatt
ttctatacgg cacccacagc gattcgggcc tttatcaaaa
tgggtgaagg catccccaat aaatatgaca tgagttccct
gcgcctctta ggaaccgtgg gtgaaccgat taacccagaa
gcttggatgt ggtaccaccg ggtcattggt ggcgaacgtt
gtcccattgt tgatacatgg tggcaaacgg aaaccggtgg
tgtgatgatt acgcctttac ccggtgcaac tcccacaaaa
cccggctcgg caactcgtcc ttttccgggg attgtggcgg
atgtcgttga ccttgatgga aattccgttg gtgacaacga
aggcggctac ctggtagtga aacaaccctg gcctgggatg
atgcgtactg tttacggcaa tcccgaacgc ttccggtcta
cctattggga gcacatcgcc ccgaaagatg gacaatacct
ttatttcgca ggtgacgggg cacgccgtga ccaagatggc
tatttttgga ttatgggtcg cgtcgatgat gtcttaaatg
tttcgggcca tcgcctcggc accatggaag tggaatcggc
cctcgtttcc caccctgccg tcgccgaagc agccgtggtt
ggaaagccag atccggttaa gggggaagag gtgtttgcct
ttgtcaccct tgagggcacc tacagtccga gcgacgatct
cgtaacggaa ctcaaggccc atgtggtgaa agaaattggg
gcgatcgccc gtccgggaga aatccgtttt gccgatgtaa
tgcccaaaac ccgttctggg aagatcatgc ggcgtttgtt
gcgaaaccta gccgcaggtc aggaaattgt gggcgacacc
tccaccctcg aagaccgcag cgtcctcgat caactccggg
gctaa
The acsA coding sequence in the exemplary organism Synechococcus sp. PCC 7002 encodes a protein included in GenBank under accession number YP—001735082.1, having the following amino acid sequence:
(SEQ ID NO: 2)
MSEQNIESIL QEQRLFSPAP DFAAEAQIKS LDQYQALYDR
AKNDPEGFWG ELAEQELEWF EKWDKVLDWQ PPFAKWFVNG
KINISYNCLD RHLKTWRKNK AALIWEGEPG DSRTLTYAQL
HHEVCQFANA MKKLGVKKGD RVGIYMPMIP EAVVALLACA
RIGAPHTVIF GGFSAEALRS RLEDAEAKLV ITADGGFRKD
KAVPLKDQVD AAIADHHAPS VENVLVVQRT KEPVHMEAGR
DHWWHDLQKE VSADCPAEPM DAEDMLFILY TSGTTGKPKG
VVHTTGGYNL YTHITTKWIF DLKDDDVYWC GADVGWITGH
SYITYGPLSN GATVLMYEGA PRPSNPGCYW EIIQKYGVTI
FYTAPTAIRA FIKMGEGIPN KYDMSSLRLL GTVGEPINPE
AWMWYHRVIG GERCPIVDTW WQTETGGVMI TPLPGATPTK
PGSATRPFPG IVADVVDLDG NSVGDNEGGY LVVKQPWPGM
MRTVYGNPER FRSTYWEHIA PKDGQYLYFA GDGARRDQDG
YFWIMGRVDD VLNVSGHRLG TMEVESALVS HPAVAEAAVV
GKPDPVKGEE VFAFVTLEGT YSPSDDLVTE LKAHVVKEIG
AIARPGEIRF ADVMPKTRSG KIMRRLLRNL AAGQEIVGDT
STLEDRSVLD QLRG
Homologs of acsA include genes or gene products encoded thereby that are homologous to the acsA gene or its product. Proteins and/or protein sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity (e.g., identity) over 50, 100, 150 or more residues (nucleotides or amino acids) is routinely used to establish homology (e.g., over the full length of the two sequences to be compared). Higher levels of sequence similarity (e.g., identity), e.g., 30%, 35% 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, can also be used to establish homology. Accordingly, homologs of the genes or gene products described herein include genes or gene products having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the genes or gene products described herein. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. The homologous proteins should demonstrate comparable activities and, if an enzyme, participate in the same or analogous pathways. “Orthologs” are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same or similar function in the course of evolution. As used herein “orthologs” are included in the term “homologs”.
For sequence comparison and homology determination, one sequence typically acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters. A typical reference sequence of the invention is a nucleic acid or amino acid sequence corresponding to acsA or other genes or products described herein.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2008)).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity for purposes of defining homologs is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. The above-described techniques are useful in identifying homologous sequences for use in the methods described herein.
The terms “identical” or “percent identity”, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described above (or other algorithms available to persons of skill) or by visual inspection.
The phrase “substantially identical”, in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90, about 95%, about 98%, or about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Such “substantially identical” sequences are typically considered to be “homologous”, without reference to actual ancestry. Preferably, the “substantial identity” exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues, at least about 250 residues, or over the full length of the two sequences to be compared.
Non-limiting examples of gene-product homologs of the acsA gene in various microorganisms include the acetyl-coenzyme A synthetase from Fischerella sp. JSC-11 (Genbank Accession No. ZP—08986431.1), the acetyl-coenzyme A from Moorea producta 3 L synthetase (Genbank Accession No. ZP—08425677.1), the acetate/CoA from Cyanothece sp. PCC 7822 ligase (Genbank Accession No. YP—003886065.1), the acetyl-CoA from Cyanothece sp. PCC 7424 synthetase (Genbank Accession No. YP—002378472.1), the unnamed protein product from Thermosynechococcus elongatus BP-1 (Genbank Accession No. NP—681677.1), the unnamed protein product from Anabaena variabilis ATCC 29413 (Genbank Accession No. YP—321725.1), the acetate-CoA ligase from Cylindrospermopsis raciborskii CS-505 (Genbank Accession No. ZP—06308209.1), the acetyl-CoA synthetase from Nostoc punctiforme PCC 73102 (Genbank Accession No. YP—001869493.1), the acetate-CoA ligase from Microcoleus chthonoplastes PCC 7420 (Genbank Accession No. ZP—05030125.1), the acetyl-coenzyme A synthetase from Nodularia spumigena CCY9414 (Genbank Accession No. ZP—01629204.1), the acetyl-CoA synthetase from Microcystis aeruginosa NIES-843 (Genbank Accession No. YP—001660936.1), the acetate/CoA ligase from ‘Nostoc azollae’ 0708 (Genbank Accession No. YP—003723268.1), the acsA gene from Microcystis aeruginosa PCC 7806 (Genbank Accession No. CAO86486.1), the acetyl-coenzyme A synthetase from Microcoleus vaginatus FGP-2 (Genbank Accession No. ZP—08490634.1), the Acetate-CoA ligase from Raphidiopsis brookii D9 (Genbank Accession No. ZP—06304063.1), the acsA gene product from Acaryochloris marina MBIC11017 (Genbank Accession No. YP—001517064.1), the acetyl-CoA synthetase from Acaryochloris sp. CCMEE 5410 (Genbank Accession No. ZP—09248274.1), the acetyl-CoA synthetase from Oscillatoria sp. PCC 6506 (Genbank Accession No. ZP—07113076.1), the acetyl-CoA synthetase from Cyanothece sp. PCC 7425 (Genbank Accession No. YP—002484565.1), the Acetate-CoA ligase from Lyngbya sp. PCC 8106 (Genbank Accession No. ZP—01623739.1), the unnamed protein product from Trichodesmium erythraeum IMS101 (Genbank Accession No. YP—722064.1), the acetyl-CoA synthetase from Arthrospira platensis str. Paraca (Genbank Accession No. ZP—06383883.1), the acetate/CoA ligase from Arthrospira maxima CS-328 (Genbank Accession No. ZP—03274675.1), the acetyl-coenzyme A synthetase from Arthrospira sp. PCC 8005 (Genbank Accession No. ZP—09782650.1), the acetate/CoA ligase from Arthrospira maxima CS-328 (Genbank Accession No. EDZ93724.1), the acetyl-coenzyme A synthetase from Arthrospira sp. PCC 8005 (Genbank Accession No. CCE18403.1), the unnamed protein product from Cyanothece sp. PCC 8802 (Genbank Accession No. YP—003138301.1), the acetate/CoA ligase from Cyanothece sp. PCC 8802 (Genbank Accession No. ACV01466.1), the acetyl-CoA synthetase from Cyanothece sp. PCC 8801 (Genbank Accession No. YP—002373634.1), the acetyl-coenzyme A synthetase from Cyanothece sp. ATCC 51472 (Genbank Accession No. ZP—08974038.1), the unnamed protein product from Synechococcus elongatus PCC 6301 (Genbank Accession No. ZP—08974038.1), the acetyl-CoA synthetase from Cyanothece sp. ATCC 51142 (Genbank Accession No. YP—001803432.1), the acetyl-coenzyme A synthetase from Cyanothece sp. CCY0110 (Genbank Accession No. ZP—01730332.1), the AMP-dependent synthetase and ligase from Crocosphaera watsonii WH 8501 (Genbank Accession No. ZP—00514814.1), the acetate-CoA ligase from Synechococcus sp. PCC 7335 (Genbank Accession No. ZP—05036109.1), the acetyl-coenzyme A synthetase from Synechococcus sp. WH 8102 (Genbank Accession No. NP—897106.1), the acetate-CoA ligase from Synechococcus sp. WH 7805 (Genbank Accession No. ZP—01123920.1), the acetate-CoA ligase from Synechococcus sp. WH 8109 (Genbank Accession No. ZP—05788236.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9313 (Genbank Accession No. NP—894222.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9303 (Genbank Accession No. YP—001017906.1), the acetyl-CoA synthetase from Synechococcus sp. WH 7803 (Genbank Accession No. YP—001224763.1), the acetyl-coenzyme A synthetase from Synechococcus sp. RS9917 (Genbank Accession No. ZP—01080065.1), the acetyl-coenzyme A synthetase from Synechococcus sp. WH 8016 (Genbank Accession No. ZP—08955323.1), the acetate-CoA ligase from Synechococcus sp. CC9311 (Genbank Accession No. YP—730758.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9211 (Genbank Accession No. YP—001550915.1), the acetate-CoA ligase from Synechococcus sp. CC9902 (Genbank Accession No. YP—377326.1), the acetate-CoA ligase from Synechococcus sp. BL107 (Genbank Accession No. ZP—01467683.1), the acetyl-coenzyme A synthetase from Synechococcus sp. RS9916 (Genbank Accession No. ZP—01471857.1), the acetyl-coenzyme A synthetase from Synechococcus sp. CC9605 (Genbank Accession No. YP—381449.1), the acetyl-coenzyme A synthetase from Synechococcus sp. CB0205 (Genbank Accession No. ZP—07971118.1), the acetyl-CoA synthetase from Synechococcus sp. RCC307 (Genbank Accession No. YP—001227601.1), the acetyl-coenzyme A synthetase from Synechococcus sp. CB0101 (Genbank Accession No. ZP—07973216.1), the acetate-CoA ligase from Cyanobium sp. PCC 7001 (Genbank Accession No. ZP—05043915.1), the acetate-CoA ligase from Synechococcus sp. WH 5701 (Genbank Accession No. ZP—01085120.1), the acs gene product from Prochlorococcus marinus subsp. marinus str. CCMP1375 (Genbank Accession No. NP—875433.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. NATL2A (Genbank Accession No. YP—291252.1), the acetyl-coenzyme A synthetase from Gloeobacter violaceus PCC 7421 (Genbank Accession No. NP—923105.1), the acetyl-coenzyme A synthetase from cyanobacterium UCYN-A (Genbank Accession No. YP—003421821.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. NATL1A (Genbank Accession No. YP—001014503.1), the acetyl-coenzyme A synthetase from Singulisphaera acidiphila DSM 18658 (Genbank Accession No. ZP—09573232.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus subsp. pastoris str. CCMP1986 (Genbank Accession No. NP—892737.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9312 (Genbank Accession No. YP—397116.1), the acetate/CoA ligase from Meiothermus ruber DSM 1279 (Genbank Accession No. YP—003507084.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9215 (Genbank Accession No. YP—001483902.1), the acs gene product from Prochlorococcus marinus str. AS9601 (Genbank Accession No. YP—001009068.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9515 (Genbank Accession No. YP—001011000.1), the acetate-CoA ligase from Prochlorococcus marinus str. MIT 9202 (Genbank Accession No. ZP—05137406.1), the acetyl-coenzyme A synthetase from Marinithermus hydrothermalis DSM 14884 (Genbank Accession No. YP—004368660.1), the acetyl-coenzyme A synthetase from Prochlorococcus marinus str. MIT 9301 (Genbank Accession No. YP—001090869.1), the unnamed protein product from Nostoc sp. PCC 7120 (Genbank Accession No. NP—488297.1), the acetate/CoA ligase from Truepera radiovictrix DSM 17093 (Genbank Accession No. YP—003703935.1), the acetate/CoA ligase from Haliangium ochraceum DSM 14365 (Genbank Accession No. YP—003269915.1), the acetyl-coenzyme A synthetase from Gemmata obscuriglobus UQM 2246 (Genbank Accession No. ZP—02733777.1), the acetyl-coenzyme A synthetase from Isosphaera pallida ATCC 43644 (Genbank Accession No. YP—004179760.1), the acetyl-CoA synthetase from Chloroherpeton thalassium ATCC 35110 (Genbank Accession No. YP—001995147.1), the acetate-CoA ligase from Planctomyces maris DSM 8797 (Genbank Accession No. ZP—01856978.1), the acetyl-CoA synthetase from Thermus thermophilus HB8 (Genbank Accession No. YP—144514.1), the acetate/CoA ligase from Planctomyces limnophilus DSM 3776 (Genbank Accession No. YP—003632128.1), the acetyl-CoA synthetase from Thermus thermophilus HB27 (Genbank Accession No. YP—004855.1), the acetyl-coenzyme a synthetase from Oceanithermus profundus DSM 14977 (Genbank Accession No. YP—004057553.1), the acetyl-coenzyme A synthetase from Candidatus Koribacter versatilis Ellin345 (Genbank Accession No. YP—592595.1), the acetate/CoA ligase from Meiothermus silvanus DSM 9946 (Genbank Accession No. YP—003684983.1), the acetate-CoA ligase from Verrucomicrobium spinosum DSM 4136 (Genbank Accession No. ZP—02931268.1), the acetate/CoA ligase from Thermus aquaticus Y51MC23 (Genbank Accession No. ZP—03496427.1), the acetyl-coenzyme A synthetase from Symbiobacterium thermophilum IAM 14863 (Genbank Accession No. YP—074710.1), the acetate/CoA ligase from bacterium Ellin 514 (Genbank Accession No. ZP—03630513.1), the acetyl-CoA synthetase from uncultured candidate division OP1 bacterium (Genbank Accession No. BAL56248.1), the acetyl-coenzyme A synthetase from Blastopirellula marina DSM 3645 (Genbank Accession No. ZP—01092728.1), the acs2 gene product from Thermus scotoductus SA-01 (Genbank Accession No. YP—004201921.1), the acetyl-coenzyme A synthetase from Archaeoglobus veneficus SNP6 (Genbank Accession No. YP—004341076.1), the Acetyl-coenzyme A synthetase from Desulfitobacterium dehalogenans ATCC 51507 (Genbank Accession No. ZP—09634500.1), the unnamed protein product from Candidatus Chloracidobacterium thermophilum B (Genbank Accession No. YP—004864177.1), the acetate-CoA ligase from Acidobacterium capsulatum ATCC 51196 (Genbank Accession No. YP—002755829.1), the acetate/CoA ligase from Pirellula staleyi DSM 6068 (Genbank Accession No. YP—003369860.1), the acetyl-CoA synthetase from Chlorobium chlorochromatii CaD3 (Genbank Accession No. YP—379980.1), the acetate-CoA ligase from Myxococcus xanthus DK 1622 (Genbank Accession No. YP—630789.1), the acetate-CoA ligase from Myxococcus fulvus HW-1 (Genbank Accession No. YP—004667083.1), the unnamed protein product from Candidatus Solibacter usitatus Ellin 6076 (Genbank Accession No. YP—829106.1), the acetyl-coenzyme A synthetase from Planctomyces brasiliensis DSM 5305 (Genbank Accession No. YP—004268501.1), the acetyl-CoA synthetase from Escherichia coli UMN026 (Genbank Accession No. YP—002415210.1), the acetyl-CoA synthetase from Escherichia coli FVEC1412 (Genbank Accession No. ZP—06646805.1), the acetyl-coenzyme A synthetase from Escherichia coli FVEC1302 (Genbank Accession No. ZP—06988121.1), the acetate-CoA ligase from Escherichia coli MS 198-1 (Genbank Accession No. ZP—07115900.1), the acetyl-CoA synthetase from Escherichia coli UMN026 (Genbank Accession No. CAR15720.1), the Acs2p from Saccharomyces cerevisiae S288c (Genbank Accession No. NP—013254.1), the acetyl CoA synthetase from Saccharomyces cerevisiae YJM789 (Genbank Accession No. EDN59693.1), the K7_Acs2p from Saccharomyces cerevisiae Kyokai no. 7 (Genbank Accession No. GAA25035.1), the acetyl CoA synthetase from Saccharomyces cerevisiae RM11-1a (Genbank Accession No. EDV09449.1), the bifunctional acetyl-CoA synthetase and propionyl-CoA synthetase from Escherichia coli str. K12 substr. W3110 (GenBank Accession No. BAE78071.1), and the acetyl-coenzyme A synthetase from Pseudomonas fulva 12-X (GenBank Accession No. YP—004473024.1), among others. The genes encoding these gene products can be found in GenBank (http://www.ncbi.nlm.nih.gov/genbank/).
Homologs of acsA and AcsA discussed in the examples include the acetyl-CoA synthetase from Synechocystis sp. PCC 6803 (sll0542; Genbank Accession No. NP—442428.1; SEQ ID NOS:3 and 4), and the unnamed protein product from Synechococcus sp. PCC 7942 (SYNPCC7942—1342; Genbank Accession No. YP—400369.1; SEQ ID NOS:5 and 6)
The organic acid-tolerant microorganism of the present invention may include any microorganism that harbors an acsA gene or homolog thereof or expresses an acsA gene product or homolog thereof that is capable of being functionally deleted to render the microorganism more tolerant of organic acids. The microorganism may be eukaryotic, such as yeast, or prokaryotic, such as bacteria or archaea. Among bacteria, gram-positive, gram-negative, and ungrouped bacteria are suitable. Phototrophs, lithotrophs, and organotrophs are also suitable. In preferred versions of the invention, the microorganism is a phototroph, such as a cyanobacterium. Preferred cyanobacteria include those selected from the group consisting of Synechococcus sp., Prochlorococcus sp., Synechocystis sp., and Nostoc sp., with particularly suitable examples of Synechococcus sp. including Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, and Synechococcus sp. PCC 7002. A benefit of phototrophs is that they require only CO2 as a carbon source and are not dependent on food-based commodities or other types of biomass for which there is a growing high demand.
Functional deletion of the acsA gene product or homolog thereof in the microorganism results in increased tolerance of the microorganism to organic acids compared to a corresponding microorganism. As used herein, “corresponding microorganism” refers to a microorganism of the same species having the same or substantially same genetic and proteomic composition as a microorganism of the invention, with the exception of genetic and proteomic differences resulting from the modifications described herein for the microorganisms of the invention. Such tolerance is with respect to any organic acid present within the organism or its growth medium, particularly those that may be present in high abundance. Non-limiting examples of organic acids to which the microorganisms of the present invention have increased tolerance include acetic acid, acrylic acid, aspartic acid, benzoic acid, butyric acid, citric acid, formic acid, fumaric acid, furan dicarboxylic acid (2,5-furandicarboxylic acid), glucaric acid, glutamic acid, heptanoic acid, hexanoic acid, 3-hydroxypropionic acid (3HP), isophthalic acid, itaconic acid, lactic acid, levoascorbic acid, levulinic acid, malic acid, octanoic acid, oxalic acid, pentanoic acid, phosphoric acid, propionic acid, pyruvic acid, succinic acid (1,4 succinic acid), and terephthalic acid, among others. The examples show various aspects of increased tolerance to exemplary organic acids 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid.
One aspect of the increased tolerance to organic acids is an increase in the minimal inhibitory concentration (MIC) of a particular organic acid compared to a corresponding microorganism. MIC is the lowest concentration of an agent that will inhibit growth of a microorganism. An MIC can be determined by titrating the agent in the growth medium of the microorganism. The lowest concentration of the agent in which the microorganism is no longer able to grow is the MIC. Methods of culturing microorganisms and of detecting their growth are well known in the art and are not discussed in detail herein. A relative increase in MIC indicates a higher tolerance to an agent and indicates that the microorganism can grow in the presence of a higher concentration of the agent. Conversely, a relative decrease in MIC indicates a lower tolerance to an agent and indicates that the microorganism can grow only in the presence of a lower concentration of the agent.
Functional deletion of the acsA gene product or homolog thereof in the microorganism confers an MIC of at least about 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 250 μM, 500 μM, 1 mM, 25 mM, 50 mM, 70 mM, 100 mM, 125 mM, or 150 mM to acrylic acid; an MIC of at least about 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 260 mM, 300 mM, 350 mM, or more to 3HP; and/or an MIC of at least about 250 μM, 500 μM, 1 mM, 50 mM, 100 mM, 200 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more to propionic acid. Such MICs occur in at least Synechococcus sp. cyanobacteria, such as Synechococcus sp. PCC 7002 and Synechococcus sp. PCC 7942, when assayed at a pH of about 8. Such MICs also occur in Synechocystis sp., such as Synechocystis sp. PCC 6803, when assayed at a pH of about 8. Such MICs also occur in any other microorganism described herein, such as Prochlorococcus sp., Nostoc sp., or others.
Another aspect of increased tolerance is increased growth rate in the presence of a certain concentration of an organic acid or an equal growth rate in the presence of an increased concentration of an organic acid compared to a corresponding microorganism.
In various aspects of the invention, functional deletion of the acsA gene product or homolog thereof in the microorganism confers at least about a 1.5-fold, a 5-fold, a 10-fold, a 15-fold, a 25-fold, a 50-fold, a 75-fold, a 100-fold, a 500-fold, a 750-fold, a 1,000-fold, 1,250-fold, a 1,500-fold, a 1,750-fold, a 2,000-fold, a 2,250-fold, a 2,500-fold, a 2,750-fold, a 3,000-fold, a 3,250-fold, or a 3,500-fold increase in tolerance against an organic acid. The organic acid to which functional deletion of the acsA gene product confers such MICs may include acrylic acid, 3HP, or propionic acid, among others. In some versions of the invention, for example, functional deletion of the acsA gene product in Synechococcus sp. PCC 7002 confers at least about a 2,800-fold increase in MIC for acrylic acid, at least about a 26-fold increase in MIC for 3HP, and at least about a 100-fold increase in MIC for propionic acid at pH of about 8 (see examples below).
The increased tolerance to organic acids conferred by functional deletion of the acsA gene product or homolog thereof renders the microorganism particularly suited for producing high amounts of organic acids, many of which have industrial utility. Accordingly, the microorganism in some versions of the invention is capable of producing an organic acid that can be isolated for industrial purposes. The microorganism may be able to naturally make the organic acid, may be genetically modified to make the organic acid, or may be genetically modified to make increased amounts of the organic acid that it already makes. Non-limiting examples of organic acids that the microorganisms of the present invention can produce include acetic acid, aspartic acid, benzoic acid, citric acid, formic acid, fumaric acid, furan dicarboxylic acid (2,5-furandicarboxylic acid), glucaric acid, glutamic acid, 3-hydroxypropionic acid (3HP), isophthalic acid, itaconic acid, lactic acid, levoascorbic acid, levulinic acid, malic acid, oxalic acid, phosphoric acid, propionic acid, pyruvic acid, succinic acid (1,4 succinic acid), and terephthalic acid, among others. In preferred versions of the invention, the microorganism is capable of making at least 3HP.
In preferred versions of the invention, the microorganism is genetically modified to enhance production of at least 3HP. This can be performed by increasing expression of a gene for any one or more of the enzymes catalyzing the various steps in a 3HP-production pathway. Non-limiting examples of suitable enzymes include pyruvate kinase, pyruvate dehydrogenase, acetyl-CoA carboxylase, malonyl-CoA reductase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, aspartate decarboxylase, and β-alanine/α-ketoglutarate aminotransferase. See
Accordingly, some microorganisms of the invention include at least one recombinant nucleic acid configured to overexpress a 3HP pathway enzyme. “Recombinant” as used herein with reference to a nucleic acid molecule or polypeptide is one that has a sequence that is not naturally occurring, has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, or both. This artificial combination can be achieved, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules or polypeptides, such as genetic engineering techniques. “Recombinant” is also used to describe nucleic acid molecules that have been artificially modified but contain the same regulatory sequences and coding regions that are found in the organism from which the nucleic acid was isolated. A recombinant cell or microorganism is one that contains a recombinant nucleic acid molecule or polypeptide. “Overexpress” as used herein means that a particular gene product is produced at a higher level in one cell, such as a recombinant cell, than in a corresponding cell. For example, a microorganism that includes a recombinant nucleic acid configured to overexpress an enzyme produces the enzyme at a greater amount than a microorganism that does not include the recombinant nucleic acid.
In a preferred version of the invention, the microorganism is manipulated to express or increase expression of malonyl-CoA reductase. In some versions, the microorganism is modified to harbor a nucleic acid derived from Chloroflexus aurantiacus that encodes a malonyl-CoA reductase gene or a homolog thereof. The Chloroflexus aurantiacus malonyl-CoA reductase gene is included in GenBank under accession number AY530019 and has the following nucleotide sequence:
SEQ ID NO: 7)
aacatcatgg tatactatac ctatcgataa ttcttcaact
aattgcataa cagaacagcg atggcgacgg gggagtccat
gagcggaaca ggacgactgg caggaaagat tgcgttaatt
accggtggcg ccggcaatat cggcagtgaa ttgacacgtc
gctttctcgc agagggagcg acggtcatta ttagtggacg
gaatcgggcg aagttgaccg cactggccga acggatgcag
gcagaggcag gagtgccggc aaagcgcatc gatctcgaag
tcatggatgg gagtgatccg gtcgcggtac gtgccggtat
cgaagcgatt gtggcccgtc acggccagat cgacattctg
gtcaacaatg caggaagtgc cggtgcccag cgtcgtctgg
ccgagattcc actcactgaa gctgaattag gccctggcgc
cgaagagacg cttcatgcca gcatcgccaa tttacttggt
atgggatggc atctgatgcg tattgcggca cctcatatgc
cggtaggaag tgcggtcatc aatgtctcga ccatcttttc
acgggctgag tactacgggc ggattccgta tgtcacccct
aaagctgctc ttaatgctct atctcaactt gctgcgcgtg
agttaggtgc acgtggcatc cgcgttaata cgatctttcc
cggcccgatt gaaagtgatc gcatccgtac agtgttccag
cgtatggatc agctcaaggg gcggcccgaa ggcgacacag
cgcaccattt tttgaacacc atgcgattgt gtcgtgccaa
cgaccagggc gcgcttgaac gtcggttccc ctccgtcggt
gatgtggcag acgccgctgt ctttctggcc agtgccgaat
ccgccgctct ctccggtgag acgattgagg ttacgcacgg
aatggagttg ccggcctgca gtgagaccag cctgctggcc
cgtactgatc tgcgcacgat tgatgccagt ggccgcacga
cgctcatctg cgccggcgac cagattgaag aggtgatggc
gctcaccggt atgttgcgta cctgtgggag tgaagtgatc
atcggcttcc gttcggctgc ggcgctggcc cagttcgagc
aggcagtcaa tgagagtcgg cggctggccg gcgcagactt
tacgcctccc attgccttgc cactcgatcc acgcgatccg
gcaacaattg acgctgtctt cgattggggg gccggcgaga
ataccggcgg gattcatgca gcggtgattc tgcctgctac
cagtcacgaa ccggcaccgt gcgtgattga ggttgatgat
gagcgggtgc tgaattttct ggccgatgaa atcaccggga
caattgtgat tgccagtcgc ctggcccgtt actggcagtc
gcaacggctt acccccggcg cacgtgcgcg tgggccgcgt
gtcatttttc tctcgaacgg tgccgatcaa aatgggaatg
tttacggacg cattcaaagt gccgctatcg gtcagctcat
tcgtgtgtgg cgtcacgagg ctgaacttga ctatcagcgt
gccagcgccg ccggtgatca tgtgctgccg ccggtatggg
ccaatcagat tgtgcgcttc gctaaccgca gccttgaagg
gttagaattt gcctgtgcct ggacagctca attgctccat
agtcaacgcc atatcaatga gattaccctc aacatccctg
ccaacattag cgccaccacc ggcgcacgca gtgcatcggt
cggatgggcg gaaagcctga tcgggttgca tttggggaaa
gttgccttga ttaccggtgg cagcgccggt attggtgggc
agatcgggcg cctcctggct ttgagtggcg cgcgcgtgat
gctggcagcc cgtgatcggc ataagctcga acagatgcag
gcgatgatcc aatctgagct ggctgaggtg gggtataccg
atgtcgaaga tcgcgtccac attgcaccgg gctgcgatgt
gagtagcgaa gcgcagcttg cggatcttgt tgaacgtacc
ctgtcagctt ttggcaccgt cgattatctg atcaacaacg
ccgggatcgc cggtgtcgaa gagatggtta tcgatatgcc
agttgaggga tggcgccata ccctcttcgc caatctgatc
agcaactact cgttgatgcg caaactggcg ccgttgatga
aaaaacaggg tagcggttac atccttaacg tctcatcata
ctttggcggt gaaaaagatg cggccattcc ctaccccaac
cgtgccgatt acgccgtctc gaaggctggt cagcgggcaa
tggccgaagt ctttgcgcgc ttccttggcc cggagataca
gatcaatgcc attgcgccgg gtccggtcga aggtgatcgc
ttgcgcggta ccggtgaacg tcccggcctc tttgcccgtc
gggcgcggct gattttggag aacaagcggc tgaatgagct
tcacgctgct cttatcgcgg ctgcgcgcac cgatgagcga
tctatgcacg aactggttga actgctctta cccaatgatg
tggccgcact agagcagaat cccgcagcac ctaccgcgtt
gcgtgaactg gcacgacgtt ttcgcagcga aggcgatccg
gcggcatcat caagcagtgc gctgctgaac cgttcaattg
ccgctaaatt gctggctcgt ttgcataatg gtggctatgt
gttgcctgcc gacatctttg caaacctgcc aaacccgccc
gatcccttct tcacccgagc ccagattgat cgcgaggctc
gcaaggttcg tgacggcatc atggggatgc tctacctgca
acggatgccg actgagtttg atgtcgcaat ggccaccgtc
tattaccttg ccgaccgcaa tgtcagtggt gagacattcc
acccatcagg tggtttgcgt tacgaacgca cccctaccgg
tggcgaactc ttcggcttgc cctcaccgga acggctggcg
gagctggtcg gaagcacggt ctatctgata ggtgaacatc
tgactgaaca ccttaacctg cttgcccgtg cgtacctcga
acgttacggg gcacgtcagg tagtgatgat tgttgagaca
gaaaccgggg cagagacaat gcgtcgcttg ctccacgatc
acgtcgaggc tggtcggctg atgactattg tggccggtga
tcagatcgaa gccgctatcg accaggctat cactcgctac
ggtcgcccag ggccggtcgt ctgtaccccc ttccggccac
tgccgacggt accactggtc gggcgtaaag acagtgactg
gagcacagtg ttgagtgagg ctgaatttgc cgagttgtgc
gaacaccagc tcacccacca tttccgggta gcgcgcaaga
ttgccctgag tgatggtgcc agtctcgcgc tggtcactcc
cgaaactacg gctacctcaa ctaccgagca atttgctctg
gctaacttca tcaaaacgac ccttcacgct tttacggcta
cgattggtgt cgagagcgaa agaactgctc agcgcattct
gatcaatcaa gtcgatctga cccggcgtgc gcgtgccgaa
gagccgcgtg atccgcacga gcgtcaacaa gaactggaac
gttttatcga ggcagtcttg ctggtcactg caccactccc
gcctgaagcc gatacccgtt acgccgggcg gattcatcgc
ggacgggcga ttaccgtgta aattctacgc cacaggaacc
actaccaaac cagcatagta agagaacgat agagacgttg
caatgcgacg tctctatcat atttccggcc ccccctagac
aaacccccac gtcttcgtgt agactagaaa caggaggctg
tatgcacgtc caacaaga
The Chloroflexus aurantiacus malonyl-CoA reductase gene product is included in GenBank under accession number AAS20429 and has the following amino acid sequence:
(SEQ ID NO: 8)
MSGTGRLAGK IALITGGAGN IGSELTRRFL AEGATVIISG
RNRAKLTALA ERMQAEAGVP AKRIDLEVMD GSDPVAVRAG
IEAIVARHGQ IDILVNNAGS AGAQRRLAEI PLTEAELGPG
AEETLHASIA NLLGMGWHLM RIAAPHMPVG SAVINVSTIF
SRAEYYGRIP YVTPKAALNA LSQLAARELG ARGIRVNTIF
PGPIESDRIR TVFQRMDQLK GRPEGDTAHH FLNTMRLCRA
NDQGALERRF PSVGDVADAA VFLASAESAA LSGETIEVTH
GMELPACSET SLLARTDLRT IDASGRTTLI CAGDQIEEVM
ALTGMLRTCG SEVIIGFRSA AALAQFEQAV NESRRLAGAD
FTPPIALPLD PRDPATIDAV FDWGAGENTG GIHAAVILPA
TSHEPAPCVI EVDDERVLNF LADEITGTIV IASRLARYWQ
SQRLTPGARA RGPRVIFLSN GADQNGNVYG RIQSAAIGQL
IRVWRHEAEL DYQRASAAGD HVLPPVWANQ IVRFANRSLE
GLEFACAWTA QLLHSQRHIN EITLNIPANI SATTGARSAS
VGWAESLIGL HLGKVALITG GSAGIGGQIG RLLALSGARV
MLAARDRHKL EQMQAMIQSE LAEVGYTDVE DRVHIAPGCD
VSSEAQLADL VERTLSAFGT VDYLINNAGI AGVEEMVIDM
PVEGWRHTLF ANLISNYSLM RKLAPLMKKQ GSGYILNVSS
YFGGEKDAAI PYPNRADYAV SKAGQRAMAE VFARFLGPEI
QINAIAPGPV EGDRLRGTGE RPGLFARRAR LILENKRLNE
LHAALIAAAR TDERSMHELV ELLLPNDVAA LEQNPAAPTA
LRELARRFRS EGDPAASSSS ALLNRSIAAK LLARLHNGGY
VLPADIFANL PNPPDPFFTR AQIDREARKV RDGIMGMLYL
QRMPTEFDVA MATVYYLADR NVSGETFHPS GGLRYERTPT
GGELFGLPSP ERLAELVGST VYLIGEHLTE HLNLLARAYL
ERYGARQVVM IVETETGAET MRRLLHDHVE AGRLMTIVAG
DQIEAAIDQA ITRYGRPGPV VCTPFRPLPT VPLVGRKDSD
WSTVLSEAEF AELCEHQLTH HFRVARKIAL SDGASLALVT
PETTATSTTE QFALANFIKT TLHAFTATIG VESERTAQRI
LINQVDLTRR ARAEEPRDPH ERQQELERFI EAVLLVTAPL
PPEADTRYAG RIHRGRAITV
Exemplary homologs of the Chloroflexus aurantiacus malonyl-CoA reductase gene product include but are not limited to the short-chain dehydrogenase/reductase SDR from Chloroflexus aggregans DSM 9485 (Genbank Accession No. YP—002462600.1), the short-chain dehydrogenase/reductase SDR from Oscillochloris trichoides DG6 (Genbank Accession No. ZP—07684596.1), the short-chain dehydrogenase/reductase SDR from Roseiflexus castenholzii DSM 13941 (Genbank Accession No. YP—001433009.1), the short-chain dehydrogenase/reductase SDR from Roseiflexus sp. RS-1 (Genbank Accession No. YP—001277512.1), among others. The genes encoding these gene products can be found in GenBank.
In some versions of the invention, the microorganism is manipulated to express or increase expression of acetyl-CoA carboxylase, either alone, with malonyl-CoA reductase, or with other enzymes. This can be performed by introducing exogenous acetyl-CoA carboxylase subunit genes into the microorganism, by introducing highly expressed promoters in front of the endogenous acetyl-CoA carboxylase subunit genes, by increasing translational efficiency, or by other means. In bacteria, acetyl-CoA carboxylase is a multisubunit enzyme that is encoded by four genes, accA, accB, accC, and accD. Exemplary acetyl-coA carboxylase subunit genes for use in the present invention can be those found in Synechococcus sp. PCC 7002 or homologs thereof. The complete genome of Synechococcus sp. PCC 7002 can be found in GenBank under Accession No. NC—010475. The gene for accA can be found at positions 2536162-2537139 of NC—010475, the gene product of which has a sequence represented by GenBank Accession No. YP—001735676.1. The gene for accB can be found at positions 60707-61204 of NC—010475, the gene product of which has a sequence represented by GenBank Accession No. YP—001733325.1. The gene for accC can be found at positions 2210473-2211819 of NC—010475, the gene product of which has a sequence represented by GenBank Accession No. YP—001735364.1″. The gene for accD can be found at positions 64484-65443 of NC—010475, the gene product of which has a sequence represented by GenBank Accession No. YP—001733331.1. Suitable promoters for increasing expression of these genes are known in the art. In some versions of the invention, an artificial operon comprising the accD, accA, accB, and accC subunits from E. coli can be introduced into the microorganism for expression or overexpression of acetyl-CoA carboxylase. See, e.g., US 2011/0165637 to Pfleger et al., which is incorporated herein by reference.
Other genetic modifications of the microorganism of the present invention include any of those described in U.S. Pat. No. 8,048,624 to Lynch, U.S. Pub. 2011/0125118 to Lynch, and U.S. Pub. 2010/0210017 to Gill et al., all of which are attached hereto. See also Warnecke et al. Metabolic Engineering (2010) 12:241-250. The genetic modifications in these references may be to enhance organic acid tolerance and/or increase organic acid production. The microorganism of the present invention may also be modified with homologs of any of the genes, constructs, or other nucleic acids discussed in the above references. Non-limiting examples of the genes that may be manipulated or introduced include tyrA, aroA, aroB, aroC, aroD, aroE, aroF, aroG, aroH, aroK, aroL, aspC, entA, entB, entC, entD, entE, entF, folA, folB, folC, folD, folE, folK, folP, menA, menB, menC, menD, menE, menF, pabA, pabB, pabC, pheA, purN, trpA, trpB, trpC, trpD, trpE, tyrB, ubiA, ubiB, ubiG, ubiD, ubiE, ubiF, ubiG, ubiH, ubiX, and ydiB, or homologs thereof. A non-limiting example of a pathway that may be manipulated includes the chorismate super-pathway. These genes and pathways are primarily but not exclusively related to the production and tolerance of 3HP.
Exogenous, heterologous nucleic acids encoding enzymes to be expressed in the microorganism are preferably codon-optimized for the particular microorganism in which they are introduced. Codon optimization can be performed for any nucleic acid by a number of programs, including “GENEGPS”-brand expression optimization algorithm by DNA 2.0 (Menlo Park, Calif.), “GENEOPTIMIZER”-brand gene optimization software by Life Technologies (Grand Island, N.Y.), and “OPTIMUMGENE”-brand gene design system by GenScript (Piscataway, N.J.). Other codon optimization programs or services are well known and commercially available.
In addition to the microorganism itself, other aspects of the present invention include methods of producing organic acids with the microorganisms of the present invention. The methods involve culturing the microorganism in conditions suitable for growth of the microorganism. The microorganism either directly produces the organic acid or acids of interest or produces organic-acid precursors from which the organic acid or acids of interest are spontaneously converted. Such conditions include providing suitable carbon sources for the particular microorganism along with suitable micronutrients. For eukaryotic microorganisms and heterotrophic bacteria, suitable carbon sources include various carbohydrates. Such carbohydrates may include biomass or other suitable carbon sources known in the art. For phototrophic bacteria, suitable carbon sources include CO2, which is provided together with light energy.
The microorganism of the present invention is capable of being cultured in high concentrations of the organic acid or acids that the organism is configured to produce. This enables increased production of the organic acid or acids of interest. The microorganism can be cultured in the presence of an organic acid in an amount up to the MIC for that organic acid. Various MICs for exemplary organic acids are described herein. Accordingly, the microorganisms of the invention (i.e., Synechococcus sp., Prochlorococcus sp., etc.) can be cultured in the presence of at least about 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 250 μM, 500 μM, 750 μM, 1 mM, 25 mM, 50 mM, 70 mM, 75 mM, 100 mM, 125 mM, or 150 mM acrylic acid; at least about 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 260 mM, 300 mM, or 350 mM 3HP; and/or at least about 250 μM, 500 μM, 750 μM, 1 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM propionic acid. Such culturing preferably occurs at a pH of about 8.
Some versions of the invention include using acsA or a homolog thereof as a counter selection marker. The acsA or homolog thereof provides sensitivity to the organic acids acrylic acid, 3HP, and propionic acid. By replacing the native copy of acsA or homolog thereof with a gene of interest through double homologous recombination, one can select for cells which have gone through the recombination event by plating on acrylic acid or another organic acid as described herein. Acrylic acid is preferred because it has the lowest MIC value and requires the lowest concentration for selection. Through this method, one can introduce a gene or operon of interest onto a chromosome without the need for antibiotics. Additionally, one can plate on a higher organic acid concentration, i.e., one closer to the MIC value of the acsA mutant strain, to cure the strain of interest of any copies of the wild type chromosome. This is of particular interest because it can be difficult to create a homozygous strain using antibiotics as the selection agent.
One version comprises using acsA or homolog thereof as a counter selection marker for introducing DNA fragments of interest into the acsA or homolog locus. An exemplary version is shown in
Another version comprises using the acsA gene or homolog thereof as a counter selection marker to introduce DNA fragments of interest into loci other than an acsA or homolog locus without leaving an antibiotic resistance marker. An exemplary version is shown in
To increase the utility of acsA as a counter selection marker, two point mutations can be made, T144C and G150C. These point mutations maintain the same amino acid sequence but break up a run of base pairs that create a loss of function mutation hot spot. By creating these mutations, the background mutation frequency of this gene is reduced. This mutant version of acsA, acsA*, can be incorporated onto a non-chromosomal plasmid, such as the endogenous plasmid pAQ1 of a ΔacsA strain of PCC 7002. This base strain allows for incorporating a gene or operon of interest onto the pAQ1 plasmid without the use of antibiotics and quickly creating a homozygous strain.
The elements and method steps described herein can be used in any combination whether explicitly described or not.
The singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.
Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
All patents, patent publications, and peer-reviewed publications (i.e., “references”) cited herein are expressly incorporated by reference to the same extent as if each individual reference were specifically and individually indicated as being incorporated by reference. In case of conflict between the present disclosure and the incorporated references, the present disclosure controls.
It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims.
One of the potential applications of metabolic engineering is the use of cyanobacteria to photosynthetically produce commodity chemicals traditionally derived from petroleum. In particular, acrylic acid has been identified as a high-value product that could be biologically derived. Unfortunately, a viable metabolic pathway has not previously been identified for its direct production.
As described in further detail below, a mutation resulting in increased tolerance to 3HP was discovered through investigating the metabolism of a sulfur compound, dimethylsulfoniopropionate (DMSP), by Synechococcus sp. PCC 7002 (PCC 7002). PCC 7002 was grown in the presence of DMSP to determine if it could be metabolized. This surprisingly resulted in the accumulation of acrylic acid, a by-product of DMSP metabolism, showing that Synechococcus sp. can produce acrylic acid. The accumulation of acrylic acid in the growth medium caused a stall in growth of the cyanobacteria, suggesting it had a toxic effect. After an additional incubation period, growth began to resume. It was originally hypothesized that the ability to grow in the presence of acrylic acid was the result of an adaptation to the stress through altered gene regulation. This hypothesis was later invalidated after an experiment was performed involving growing “unadapted” cells on solid medium containing acrylic acid. The number of colonies on the plate relative to a control suggested that a loss of function mutation was occurring that resulted in the ability to grow in the presence of acrylic acid. Additionally, all mutants obtained through growth in the presence of acrylic acid had increased tolerance levels to 3HP. The increase in tolerance caused by the mutation resulted in a strain of cyanobacteria constituting a platform for either 3HP or acrylic acid production.
Steps were taken to identify the site of the mutation. An RNA sequencing experiment was performed to characterize differential gene expression in the presence of either DMSP or acrylic acid. This data set was used to identify genes that had single base pair mutations relative to the wild type strain. Through this analysis, mutations were identified in the gene acsA. In order to determine if acsA was involved in acrylic acid and 3HP toxicity, a strain of PCC 7002 was created that had a deletion of the acsA gene. This strain, PCC 7002 ΔacsA, had increased MIC values compared to wild type PCC 7002. These experiments determined that it is a loss of function of acsA that results in increased tolerance. The gene acsA was annotated as an acetyl-CoA ligase.
In order to demonstrate the utility of the ΔacsA strain, a pathway for producing 3HP was introduced into both the wild type PCC 7002 and ΔacsA strains. Several pathways exist for the production of 3HP from central metabolites. The chosen pathway involves an enzyme from the CO2 fixation pathway of the thermophilic bacterium Chloroflexus aurantiacus. In this pathway, malonyl-CoA is converted to 3HP through a two-step reaction catalyzed by the enzyme malonyl-CoA reductase. Results have shown that expression of malonyl-CoA reductase confers the ability to produce 3HP on the order of 50 μM.
The result of these experiments is an engineered strain of PCC 7002 that can produce 3HP and is more tolerant to 3HP than wild type PCC 7002 or other cyanobacterial species. Further work will increase the yield of 3HP. The approach to increasing yield will involve further metabolic engineering and optimizing of culturing conditions. To further engineer this strain, expression of the malonyl-CoA reductase will be optimized and genes related to making malonyl-CoA will be over-expressed. Additionally, the current and further engineered strains will be cultured in a photobioreactor in order to monitor 3HP production under optimal growth conditions, and culture parameters will be adjusted to increase yields. The outcome of this work will be a strain of cyanobacteria with optimized culturing conditions that will result in a competitive yield of 3HP.
Background and Significance of Examples
Engineering Bacteria to Produce Commodity Chemicals
A current focus of metabolic engineering and synthetic biology is the development of new methods for producing commodity chemicals that are traditionally produced from petroleum [1,2]. Demand for methods of bioconversion of renewable resources (biomass or CO2) to these compounds has increased due to price volatility and reliance on foreign production of oil, concerns of increasing atmospheric CO2, and increased consumer demand for “green” and sustainable products. An example of recent commercial success is the production of 1,3-propanediol (a precursor of nylon-like materials) by DuPont via Escherichia coli fermentation of corn sugar [3].
Another compound that could be produced from renewable sources is acrylic acid. Acrylic acid, traditionally produced through the oxidation of propene, is used in coatings, finishes, plastics, and superabsorbent polymers [4]. US demand for acrylic acid continues to grow, exceeding 1×109 kg/year, and is outpacing current production [4]. For this reason, non-petroleum based, sustainable methods for producing acrylic acid would be of value. Unfortunately, a thermodynamically favorable pathway for complete biological production of acrylic acid has not been identified [5]. An alternative route would be biological production of 3-hydroxypropionic acid (3HP), followed by a non-biological catalytic conversion to acrylic acid. Additionally, 3HP can be converted to other commodity chemicals including acrylamide and 1,3-propanediol [6]. One company, OPX Biotechnologies, has developed a bio-based technology for producing acrylic acid, via Escherichia coli fermentation of sugars to 3HP [7].
Cyanobacteria as an Alternative to Heterotrophic Bacteria
One of the concerns of using heterotrophic bacteria and yeast for fuel and chemical production is the use of food based commodities as feedstock. As the global population continues to grow and the cost of agricultural commodities continues to rise, an alternative route for biological production of commodity chemicals may be needed. An attractive alternative is to use cyanobacteria to convert CO2 and light energy directly into chemical products. Using CO2 rather than organic carbon as an input circumvents the problem of using agricultural commodities and could potentially decrease costs. Species of cyanobacteria are susceptible to genetic modification and have well studied metabolisms [8,9]. Recently, cyanobacteria have been engineered to produce a variety of chemicals and fuels including ethanol, hydrogen, isobutyraldehyde, isoprene, sugars, and fatty acids [10-14].
In order for cyanobacteria to be effective host systems for chemical production, they will have to produce the compound of interest in high titers and have improved resistance to end product toxicity. As presented below, a mutant strain of cyanobacteria was isolated with dramatically increased tolerance to acrylic acid and 3HP. This mutation was identified through exploring the role cyanobacteria play in metabolism of the marine sulfur compound dimethylsulfoniopropionate (DMSP).
Metabolism of the Sulfur Compound DMSP
DMSP is an organic sulfur compound produced by eukaryotic algae and plants that accounts for 1-10% of primary productivity in the oceans [16]. DMSP has been shown to act as an osmoprotectant, antioxidant, predator deterrent, and a sink for reduced sulfur in marine eukaryotic algae [17,18]. Upon its release into the water, DMSP is metabolized by bacterioplankton for use as a carbon and reduced sulfur source [19]. The catabolism of DMSP has the potential to supply 1-15% of total carbon demand and nearly all of the sulfur demand for these bacterial communities [20]. Additionally, cyanobacteria have been shown to account for 10-34% of total DMSP assimilation in light-exposed waters [21, 22].
DMSP is broken down through two major pathways. These pathways involve either direct cleavage of DMSP into dimethylsulfide (DMS) and acrylic acid or an initial demethylation followed by a cleavage reaction to form methanethiol and acrylic acid [16, 23-25]. Methanethiol is then used as a reduced sulfur source in methionine biosynthesis, while acrylic acid can be further metabolized into 3HP and used as a carbon source [26,27]. Additionally, release of DMS into the atmosphere from marine waters has been identified as a key intermediate in the cycling of terrestrial and marine sulfur pools [28]. While several genes have been identified in DMSP metabolism, none have been found in cyanobacteria.
Recent studies have shown that two different groups of cyanobacteria are involved in the metabolism of DMSP. These studies demonstrated that both Synechococcus and Prochlorococcus species are capable of assimilating radio labeled DMSP and methanethiol. In addition, four pure strains of Synechococcus were analyzed for DMSP assimilation. Two of the four strains were able to transport and assimilate DMSP, while another produced DMS [22]. Of the species of cyanobacteria currently being used in metabolic engineering, only one, Synechococcus sp. PCC 7002, is found in marine environments and potentially exposed to DMSP.
Metabolism of DMSP can result in the accumulation of several metabolites, including acrylic acid and 3HP, and may alter growth patterns due to its use as a carbon and sulfur source. PCC 7002 was cultured in the presence of 5 mM DMSP and analyzed for the presence of acrylic acid and 3HP. Growth was determined by monitoring OD730 while metabolic byproducts were measured through high pressure liquid chromatography (HPLC) and gas chromatography (GC). During incubation with DMSP, an increase in OD730 similar to a control culture was observed for several doubling events, followed by a delay in increased OD730 (
Accumulation of organic acid anions in the cytoplasm of bacteria has been shown to block metabolic pathways and arrest growth [32,33]. In addition to blocking metabolic pathways, high concentrations of organic acids have been shown to reduce the proton motive force through dissociation across the membrane [34]. Because of this, the toxicity of organic acids generally increases with the hydrophobicity of the compound [35]. The minimum inhibitory concentrations (MIC) for PCC 7002, Synechococcus sp. PCC 7942, and Synechocystis sp. PCC 6803 were determined for acrylic acid, 3HP, and propionic acid at a pH of about 8 (Table 1). In all three species, acrylic acid was significantly more toxic than propionic acid, which was more toxic than 3HP. Furthermore, the toxicity of acrylic acid (pKa 4.35) to PCC 7002 was shown to be pH dependent, with toxicity increasing with decreasing pH. The low MIC for acrylic acid explains why cultures grown with DMSP become growth inhibited. Cultures with DMSP only show growth inhibition when the accumulating acrylic acid concentration reaches inhibitory concentrations. This suggests that acrylic acid and not DMSP causes the inhibition of growth. The eventual increase in OD730 suggests that mutations can arise to overcome this inhibition.
TABLE 1
Minimum inhibitory concentration of organic acids in three cyanobacteria.
Minimum inhibitory concentration is defined as the concentration
at which no increase in OD730 was observed. 1Strain A+ was isolated
from an agar plate containing 5 mM acrylic acid.
Species
Acrylic Acid
3HP
Propionic Acid
Synechococcus sp. PCC 7942
3 μM
2 mM
250 μM
Synechocystis sp. PCC 6803
50 μM
No Data
250 μM
Synechococcus sp. PCC 7002
25 μM
10 mM
4 mM
1PCC 7002 A+
7 μM
No Data
No Data
When a dense culture of PCC 7002 was plated onto solid medium containing acrylic acid, colonies resulting from spontaneous mutants uninhibited by acrylic acid were observed. The mutation frequency when selecting for growth on 50 μM acrylic acid was 7×10−6. When selecting for growth on 5 mM acrylic acid, the mutation frequency was 4×10−6. The mutation frequency is the frequency that a mutant with a given phenotype is found within the population of a culture. For example a mutation frequency of 1×10−6 suggests that in a population of 1×108 cells, there are 100 mutants. The observed mutation frequencies are suggestive of a loss of function mutation. All mutants obtained from medium containing 50 μM acrylic acid were able to grow on 5 mM acrylic acid. In addition, these colonies were able to grow in media containing concentrations of propionic acid and 3HP that were above the WT PCC 7002 MIC values. One of the mutants, PCC 7002 A+, was analyzed to determine to what degree the tolerance to organic acids had increased. MIC values for this strain are presented in Table 1. Tolerance to acrylic acid increased about 280-fold over WT PCC 7002 MIC values. Increased tolerance to 3HP and propionic acid was also observed (data not shown). Due to the increased tolerance to all three organic acids, the mutation may affect a gene that links the metabolism of acrylic acid, 3HP, and propionic acid.
In addition to looking at gene expression levels, the results from the RNA-sequencing experiment were used to identify mutations that resulted in increased tolerance to acrylic acid. An analysis for single nucleotide permutations (SNP) on the data set for each condition was performed. In order to identify potential mutation candidates, two basic assumptions were made. First, growth in cultures containing DMSP and acrylic acid would require the same mutation. Second, the mutation is a base pair change, not a deletion or insertion. From the SNP analysis, mutations in five candidate genes were identified. One of these candidates was annotated as an acetyl-CoA ligase (acsA). The mutation resulted in the change of a highly conserved tryptophan residue to a leucine (W49L) in Synechococcus sp. PCC 7002. The mutation changes an FWGE amino acid sequence in Synechococcus sp. PCC 7002 to an FLGE amino acid sequence. This mutation was a result of a G146T substitution in the acsA coding sequence. The mutation was present in ˜60% of reads that aligned to this segment of the open reading frame in both the DMSP and acrylic acid cultures. Manual inspection of control alignment data determined that this allele was only present in cultures containing DMSP and acrylic acid. The correlate of W49 is conserved in the acsA of Escherichia coli (GenBank NP—418493.1) and Bacillus subtilis (GenBank NP—390846.1), among others, suggesting it is integral to a functional protein See, e.g., Table 2.
TABLE 2
Conservation of W49 and surrounding residues of Synechococcus sp.
PCC 7002 acsA in acetyl-CoA ligases of E. coli K12 and P. fulva
Organism
Gene
Protein Sequence
SEQ ID NO:
Synechococcus sp.
acsA
F-W-G-E
Residues 48-51
PCC 7942
of SEQ ID NO: 2
E. coli K12
Acetyl-CoA ligase
F-W-G-E
Residues 39-41
of SEQ ID NO: 9
P. fulva
Acetyl-CoA ligase
F-W-G-E
Residues 38-41
of SEQ ID NO: 10
The W49L mutation residue resulted in an insoluble protein (data not shown) and, therefore, a non-functional protein. These data led to the hypothesis that loss of function of acsA would result in the observed increase in organic acid tolerance.
Without being limited by mechanism, it was hypothesized that the AcsA acetyl-CoA ligase may have a substrate specificity that would allow it to add a coenzyme A (CoA) to all three organic acids, and that the CoA bound acids or downstream metabolism of these CoA bound acids caused toxicity.
This hypothesis was tested by creating a knockout mutant of the acsA gene. This knockout was created by transforming wild type PCC 7002 with a DNA construct that would replace the acsA gene with an antibiotic resistance marker through homologous recombination. The resulting mutant, ΔacsA, was challenged with concentrations of acrylic acid, 3HP, and propionic acid above WT PCC 7002 MIC levels. In each case the ΔacsA mutant was able to grow without inhibition, including in the presence of >500 mM 3HP. Additionally, the ΔacsA mutant did not show any growth defects relative to wild type. These results show that loss of function of the acyl-CoA ligase increases the tolerance of PCC 7002 to acrylic acid and 3HP.
To confirm this phenotype is the result of the deletion mutation, a complementation mutant was created by integrating a copy of acsA into a plasmid native to PCC 7002 ΔacsA. A corresponding mutant harboring a copy of acsA-W49L was also constructed. In the presence of acrylic acid, no strains harboring wild-type acsA were capable of growing while those harboring the mutant acsA were able to grow (
In addition, the acsA gene was heterologously expressed in E. coli for protein purification and the substrate specificity was determined for AcsA in vitro (see below).
From these results, several conclusions can be drawn. DMSP is converted to acrylic acid by PCC 7002. Spontaneous mutations occur within the population that results in a drastically increased tolerance to acrylic acid, 3HP, and propionic acid. One mutation that can result in this phenotype is a loss of function or deletion of the acsA gene, which codes for an acetyl-CoA ligase.
Deletion and complementation studies were performed in various Synechococcus spp. and Synechocystis spp. The results are shown in Table 3. Replacement of the gene acsA in Synechococcus sp. PCC 7002 with an antibiotic resistance marker (aadA) resulted in a dramatic increase in tolerance to acrylic acid, 3-hydroxypropionic acid (3HP), and propionic acid. An identical level of increase was observed when acsA was replaced with a 20 base-pair barcode sequence. This phenotype was complemented in an acsA deletion strain by expression of acsA under the native promoter in another locus on the chromosome (glpK). Complementation resulted in the restored sensitivity to both acrylic acid and 3HP. The phenotype was only partially complemented upon expression of acsAW49L from the glpK locus, showing that the AW49L mutation does not result in a complete loss of AcsA activity.
Homologous genes were identified in the cyanobacteria Synechocystis sp. PCC 6803 (sll0542; SEQ ID NOS:3 and 4) and Synechococcus sp. PCC 7942 (SYNPCC7942—1342; SEQ ID NOS:5 and 6). Replacement of the gene sll0542 in PCC 6803 with an antibiotic resistance marker resulted in an increase in tolerance to acrylic acid similar to the deletion of acsA in PCC 7002. When selecting for growth of Synechocystis sp. PCC 6803 on 50 μM acrylic acid, the mutation frequency was 2×10−6.
TABLE 3
Minimum inhibitory concentrations of organic acids
acrylic acid
3-HP
Propionic acid
Species
(mM)
(mM)
(mM)
Synechococcus sp. PCC 7942
0.003
2
0.25
Synechocystis sp. PCC 6803
0.050
>35
0.25
PCC 6803 sll0542::KmR
70
<50
No Data
Synechococcus sp. PCC 7002
0.025
10
4
PCC 7002 acsA::aadA
70
260
>400
PCC 7002 acsA::BC*
70
260
No Data
PCC 7002 acsA:BC
0.015
15
No Data
glpK::acsA aadA)
PCC 7002 acsA::BC
7
No Data
No Data
glpK::acsAW49L aadA)
*BC, 20 base-pair barcode
The tolerance of PCC 7002 to acrylic acid and 3HP was dramatically increased by the deletion of the acetyl-CoA ligase gene (acsA). To obtain information regarding the AcsA-dependent toxicity, the substrate specificity of AcsA was determined.
Acyl-CoA ligase purification: Escherichia coli BL21 containing plasmid pET28b with acsA were grown in 50 mL of LB to an OD600 nm of 0.6 and induced with 1 mM IPTG. The induced culture was shaken at 37° C. for 3 hrs. The culture was centrifuged and the resulting cell pellet was frozen at −20° C. The cell pellet was processed with Novagen BugBuster Protein Extraction Reagent (Part No. 70584-3). The resulting soluble protein fraction was used for His-tag purification using Ni-NTA agarose beads (Qiagen) and Pierce 0.8-mL centrifugation columns (Part No. 89868). Washes were done with 50 mM NaH2PO4, 300 mM NaCl, and 30 mM imidazole pH 8.0. The his-tagged protein was eluted with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole pH 8.0. The insoluble fraction from the protein extraction was washed twice with BugBuster reagent followed by incubation with 400 μL 8M urea, 100 mM Tris-HCl, and 100 mM β-mercaptoethanol pH 8.2 for 30 min. The resulting solution was centrifuged at 16,000×g and the supernatant was collected. Protein fractions were run on a SDS-PAGE gel. His-tag purified protein fractions used in the acyl-CoA ligase assay were concentrated and buffered exchanged using an Amicon Ultra-4 centrifugation column. The buffer used for enzyme storage contained 0.1M NaH2PO4, 1 mM EDTA, and 10% v/v glycerol.
Acyl-CoA ligase activity assay: Acyl-CoA ligase activity was determined by measuring the loss of free Coenzyme A (CoA) over time using Ellman's reagent. (Riddles P W, Blakeley R L, & Zerner B (1979) Ellman's reagent: 5,5′-dithiobis(2-nitrobenzoic acid)—a reexamination. Analytical Biochemistry 94(1):75-81.) The enzyme reaction contained 10 mM ATP, 8 mM MgCl2, 3 mM CoA, 0.1 M NaH2PO4, 1 mM EDTA, and 2 mM of the organic acid species. The concentration of AcsA in the reaction was 500 nM. Relative activity was determined by the amount of CoA consumed in 4 min relative to an acetate control. As shown in
The sensitivity of PCC 7002 to acrylic acid due to the activity of AcsA allows for one to directly integrate DNA fragments into the acsA locus and select for acrylic acid tolerance. This method results in integration into the PCC 7002 without the use of an antibiotic resistance marker. The use of antibiotic resistance markers is limited by the number of markers available and their tendency to result in heterozygous strains. PCC 7002 carries between 4-6 copies of the chromosome and the use of resistance markers can result in strains with a mixture of native and modified chromosomes. Use of acsA as a counter-selection marker can quickly produce homozygous strains.
The acsA gene was used as a counter-selection marker to introduce DNA fragments of interest into the acsA loci on the chromosome, thereby deleting acsA without leaving an antibiotic resistance marker. Wild type PCC 7002 was transformed with barcode DNA or DNA encoding yellow fluorescent protein (YFP), each flanked with 500 base-pair sequences homologous to regions directly 5′ and 3′ of acsA. The transformed culture was then plated on 50 μM acrylic acid. Colonies appeared after 3 days. The colonies were patched onto plates containing 50 μM acrylic acid and screened for the presence of the sequence of interest. Integration of the various sequences resulted in 30-50% of colonies being positive integrations. See
The acsA gene was also used as a counter selection marker to introduce DNA fragments of interest into other loci on the chromosome without leaving an antibiotic resistance marker. In an acsA deletion strain of PCC 7002, acsA along with an antibiotic resistance marker was introduced onto the chromosome into the gene glpK. See, e.g., PCC 7002 acsA:BC glpK::acsA aadA in Table 3. glpK was used as an insertion site because it is a pseudogene in PCC 7002 due to a frameshift mutation. The acsA-resistance marker was then replaced with yellow fluorescent protein (YFP) under the expression of a constitutive promoter. This resulted in a strain of PCC 7002 with YFP integrated onto the chromosome without a residual marker. YFP expressed from the glpK locus was shown to have an equal level of expression to YFP expressed from the acsA locus. See
While the ultimate goal is to produce acrylic acid through a single biological catalyst, no complete pathway has previously been demonstrated [5]. As an alternative, 3HP can be biologically derived and then catalytically converted to acrylic acid. A 3HP production pathway was introduced into PCC 7002 ΔacsA and its ability to produce 3HP from CO2 and light energy was analyzed.
Express a Malonyl-CoA Reductase in PCC 7002
The Strain Will be Engineered to Increase Titers
Several strategies can be employed to increase 3HP production. These include altering the expression of the malonyl-CoA reductase by changing the promoter, introducing additional copies onto the plasmid, and/or codon-optimizing the gene. Codon optimization will ensure that no rare codons exist in the coding sequence that would stall translation. Additionally, flux through this pathway can be increased by introducing highly expressed promoters in front of the acetyl-CoA carboxylase genes, thus increasing the pool of malonyl-CoA. Furthermore, a genome scale metabolic model can be used to predict genetic modifications that would provide additional flux through this pathway and correct cofactor imbalances [47]. These strategies will potentially increase titers of 3HP to be comparable with production systems using heterotrophic bacteria.
Conclusions from Examples
Increasing the tolerance of the cyanobacterium Synechococcus sp. PCC 7002 to the commodity chemicals acrylic acid and 3HP and increasing at least 3HP production through metabolic engineering make biological synthesis of these compounds from CO2 a viable option.
Begemann, Matthew Brett, Pfleger, Brian Frederick
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